Biofyzikální ústav

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2019-08-26
09:04
Image Analysis of Gene Locus Positions Within Chromosome Territories in Human Lymphocytes
Stepka, K. ; Falk, Martin
One of the important areas of current cellular research with substantial impacts on medicine is analyzing the spatial organization of genetic material within the cell nuclei. Higher-order chromatin structure has been shown to play essential roles in regulating fundamental cellular processes, like DNA transcription, replication, and repair. In this paper, we present an image analysis method for the localization of gene loci with regard to chromosomal territories they occupy in 3D confocal microscopy images. We show that the segmentation of the territories to obtain a precise position of the gene relative to a hard territory boundary may lead to undesirable bias in the results; instead, we propose an approach based on the evaluation of the relative chromatin density at the site of the gene loci. This method yields softer, fuzzier ´boundaries´, characterized by progressively decreasing chromatin density. The method therefore focuses on the extent to which the signals are located inside the territories, rather than a hard yes/no classification.

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2019-08-26
09:04
Utilization of Catalytic Hydrogen Evolution in Electrochemical Analysis of Nucleic Acids
Fojta, Miroslav ; Daňhel, Aleš ; Špaček, Jan ; Havran, Luděk ; Šebest, Peter ; Orság, Petr ; Pivoňková, Hana ; Vosáhlová, J. ; Schwarzová-Pecková, K.
Catalysis of hydrogen evolution (CHE) at mercury in the presence of proteins was discoverd shortly after the introduction of polarography. In contrast, unmodified nucleic acids have not been reported to produce distinct signals due to the CHE to date. Chemically modified nucleic acids bearing certain extrinsic groups produce analytically useful signals due to hydrogen evloution catalyzed by the respective modifications. These species include (a) transition metal complexes, and (b) non-metal catalytically active organic moieties. In addition, the CHE has been reported to be invoved in guanine reduction process at the mercury-based electrodes.

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2019-08-26
09:04
2D Condensation of 5-Fluorocytosine in Different Electrolytes
Fojt, Lukáš ; Fojta, Miroslav
Two-dimensional (2D) condensation and adsorption of 5-fluorocytosine on hanging mercury drop electrode in differnt electrolytes was investigated. We have used different sodium halide salts as basic electrolytes. The influence of electrolytes composition was displayed using differential capacitance measurements. Effects of ions present in used electolytes solution was remarkable. We observed huge change in 2D condensed layers in dependence on ions type.

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2019-07-25
09:13
Use of Monovalent Copper for Sensitive Detection of Methyl Derivatives of Xanthine
Navrátil, R. ; Motlova, D. ; Jelen, F. ; Trnková, L.
This work is concerned with the electroanalysis of xanthine (Xan) and its methylated derivatives (1-, 3-, 7-, and 9-mXan) on a pencil graphite electrode in the presence of copper ions. The main idea of using copper consists in the in situ formation of the complex Cu(I)-mXan on the electrode surface and improvement of oxidative signals of the corresponding mXan during the detection. Linear sweep voltammetry (LSV) in connection with adsorption stripping techniques was used for the determination of mXan. To enhance the oxidation signals the elimination voltammetric procedure (EVP) was applied.

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2019-07-25
09:13
A Preliminary Study of Modification of 7-Deazaadenine with a Complex of Osmium Tetroxide with 2,2 '-Bipyridine
Vítová, Lada ; Havran, Luděk ; Fojta, Miroslav ; Šedo, O. ; Zdráhal, Z. ; Vespalec, Radim
Reactivity of a purine nucleic base analogue 7-deazaadenine (A*) in short oligodeoxynucleotide (ODN) towards an osmium tetroxide complex with 2,2'-bipyridine (Os, bipy) was investigated by means of electrochemistry, capillary electrophoresis and MALDI-TOF mass spectrometry. Electrochemical measurement, electrophoretic analyses and mass spectrometric analyses of reaction mixture proved that ODN with an A* residue yields an osmium adduct with properties similar to those exhibited by the well-known adduct of thymine.

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2019-07-25
09:13
Enzymatic Incorporation of Biotin into DNA for DNA Hybridization Analysis and for Sensitive Detection of PCR-Amplified DNA
Špaček, Jan ; Zenka, Martin ; Haroniková, Lucia ; Havran, Luděk ; Fojta, Miroslav
We present two enzymatical electrochemical assays for DNA analysis. For hybridization analysis we used probes with biotin-14-dC introduced to 3' OH end by terminal transferase. For detection of PCR products we used Deep Vent polymerase to incorporate biotin-14-dCduring PCR. In both cases streptavidin-alkaline phosphatase conjugate was subsequently attached to the incorporated biotins and was used to catalyze dephosphorylation of 1-naphthyl phosphate to 1-naphthol, the electrochemical signal of which was utilized for detection. Compared to the former method, biotin incorporation during PCR offers lower molar detection limits, whereas application of the biotin-tailed probe can provide us with more selective detection.

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2019-07-25
09:13
Detection of Single Nucleotide Polymorphisms Using Selective Incorporation of Biotin in DNA Strand and Subsequent Enzymatic Detection at Pencil Electrode
Plucnara, Medard ; Ecsin, E. ; Erdem, A. ; Fojta, Miroslav
Enzyme-linked electrochemical assay using DNA labeling by biotin followed by binding of enzyme-streptavidin conjugates has been found to be a potent tool for DNA diagnostic. This approach brings a special advantage of signal amplification due to the fact, that only very small number of enzymatic labels can produce a number of molecules of an electrochemically detectable product from an inactive substrate to obtain sufficiently strong signal. A new way of using of this technique combined with selective primer extension reaction designed for the detection of single nucleotide polymorphism is presented here. The assay was combined with measurements at a pencil graphite electrode, which is a very practical tool for potential clinical applications due to its cheapness and disposability.

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2019-07-25
09:13
Electrochemical Detection of p53 Protein Interactions with Plasmid DNAs Modified with Cisplatin Using Immunoprecipitation at Magnetic Microbeads
Pivoňková, Hana ; Tichý, Vlastimil ; Orság, Petr ; Šebest, Peter ; Fojta, Miroslav
Antineoplastic drug [cis-diamminedichloroplatinum(II)] (cisplatin) forms covalent adducts with DNA. Cisplatin-modified DNA can be determined sensitively using square-wave voltammetry at mercury electrodes. Tumor suppressor protein p53 binds to DNA in different modes, including sequence-and structure-specific ones and these interactions are influenced by modification of the DNA with cisplatin. In this contribution we present a simple immunoprecipitation technique with magnetic beads, followed by voltammetric determination of recovered cisplatinated DNA, for the evaluation of p53 protein binding to DNAs containing various target sites differing in their proneness to being internally modified with the platinum complex.

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2019-07-25
09:13
THE WAYS OF ACTIVATING TP53
Polášková, Alena ; Helma, Robert ; Adámik, Matěj ; Hronešová, L. ; Holacka, K. ; Ballová, L. ; Brázdová, Marie
P53, AGENTS, CELLS,P53, AGENTS, CELLS,P53, AGENTS, CELLS,P53, AGENTS, CELLS

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2019-07-25
09:13
DNA POLYMERASE STOP ASSAY FOR DETECTION OF G-QUADRUPLEXES
Petr, Marek ; Bažantová, Pavla ; Adámik, Matěj ; Kejnovská, Iva ; Dvořáková, Zuzana ; Vorlíčková, Michaela ; Pečinka, Petr ; Brázdová, Marie
In this work we performed DNA polymerase stop assays while using template DNA derived from promoter regions of VEGF and c-Myc proto-oncogenes under variety of experimental conditions. Partial stopping of DNA synthesis along the template strand was observed in the presence of K+ ions due to formation of stable G-quadruplex structures. In contrast, Na+ ions alone were unable to stabilize G-quadruplexes to stop the reaction at their site. These data suggest that K+ and Na+ ions, which are both known to stabilize G-quadruplexes, do this in different manner or extent.

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